RESUMO
Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. J. Lipid Res 2017. 58: 1439-1452.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Cisplatino/efeitos adversos , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Ceramidas/metabolismo , Córtex Renal/irrigação sanguínea , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Camundongos , Ratos , Traumatismo por Reperfusão/metabolismoRESUMO
Chronic alcohol exposure is a clinically important risk factor for the development of acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). However, the mechanisms by which alcohol sensitizes the lung to development of this disease are poorly understood. We determined the role of the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1) in alcohol enhancement of experimental endotoxin-induced ALI. Wild-type, PAI-1-/-, and integrin ß3-/- mice were fed ethanol-containing Lieber-DeCarli liquid or a control diet for 6 weeks, followed by systemic LPS challenge. LPS administration triggered coagulation cascade activation as evidenced by increased plasma thrombin-antithrombin levels and pulmonary fibrin deposition. Ethanol-exposed animals showed enhanced PAI-1 expression and pulmonary fibrin deposition with coincident exaggeration of pulmonary inflammatory edematous injury. PAI-1 deficiency markedly reduced pulmonary fibrin deposition and greatly reduced inflammation and injury without impacting upstream coagulation. Interestingly, pulmonary platelet accumulation was effectively abolished by PAI-1 deficiency in ethanol/LPS-challenged mice. Moreover, mice lacking integrin αIIBß3, the primary platelet receptor for fibrinogen, displayed a dramatic reduction in early inflammatory changes after ethanol/LPS challenge. These results indicate that the mechanism whereby alcohol exaggerates LPS-induced lung injury requires PAI-1-mediated pulmonary fibrin accumulation, and suggest a novel mechanism whereby alcohol contributes to inflammatory ALI by enhancing fibrinogen-platelet engagement.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Etanol/efeitos adversos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Lesão Pulmonar Aguda/complicações , Lesão Pulmonar Aguda/prevenção & controle , Animais , Plaquetas/metabolismo , Fibrina/metabolismo , Transtornos Hemorrágicos/complicações , Transtornos Hemorrágicos/patologia , Integrina beta3/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Inibidor 1 de Ativador de Plasminogênio/deficiência , Edema Pulmonar/complicações , Edema Pulmonar/patologia , Edema Pulmonar/prevenção & controleRESUMO
The extracellular matrix (ECM) consists of diverse components that work bidirectionally with surrounding cells to create a responsive microenvironment. In some contexts (e.g., hepatic fibrosis), changes to the ECM are well recognized and understood. However, it is becoming increasingly accepted that the hepatic ECM proteome (i.e., matrisome) responds dynamically to stress well before fibrosis. The term "transitional tissue remodeling" describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these changes in ECM may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM in liver injury are poorly understood. The goals of this work were to validate analysis of the ECM proteome and compare the impact of 6 weeks of ethanol diet and/or acute lipopolysaccharide (LPS). Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by solubility. Extracted proteins were identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Both ethanol and LPS dramatically increased the number of matrisome proteins â¼25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes. CONCLUSION: An extraction method to enrich the hepatic ECM was characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more-dramatic fibrotic changes to the liver occur. The changes to the mastrisome may contribute, at least in part, to the pathological responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. Nevertheless, there were responses that were unique to the individual and combined exposures. (Hepatology 2017;65:969-982).
Assuntos
Etanol/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Lipopolissacarídeos/farmacologia , Cirrose Hepática/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/efeitos dos fármacos , Cirrose Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Proteoma/genética , Distribuição Aleatória , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
Cisplatin, a chemotherapeutic used for the treatment of solid cancers, has nephrotoxic side effects leading to acute kidney injury (AKI). Cisplatin cannot be given to patients that have comorbidities that predispose them to an increased risk for AKI. Even without these comorbidities, 30% of patients administered cisplatin will develop kidney injury, requiring the oncologist to withhold or reduce the next dose, leading to a less effective therapeutic regimen. Although recovery can occur after one episode of cisplatin-induced AKI, longitudinal studies have indicated that multiple episodes of AKI lead to the development of chronic kidney disease, an irreversible disease with no current treatment. The standard mouse model of cisplatin-induced AKI consists of one high dose of cisplatin (>20 mg/kg) that is lethal to the animal 3 days later. This model does not accurately reflect the dosing regimen patients receive nor does it allow for the long-term study of kidney function and biology. We have developed a repeated dosing model whereby cisplatin is given once a week for 4 wk. Comparison of the repeated dosing model with the standard dosing model demonstrated that inflammatory cytokines and chemokines were induced in the repeated dosing model, but levels of cell death were lower in the repeated dosing model. The repeated dosing model had increased levels of fibrotic markers (fibronectin, transforming growth factor-ß, and α-smooth muscle actin) and interstitial fibrosis. These data indicate that the repeated dosing model can be used to study the AKI to chronic kidney disease progression as well as the mechanisms of this progression.
Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Modelos Animais de Doenças , Rim/efeitos dos fármacos , Nefroesclerose/induzido quimicamente , Animais , Antineoplásicos/administração & dosagem , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Cisplatino/administração & dosagem , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibrose , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Masculino , Camundongos , Nefroesclerose/mortalidadeRESUMO
Cisplatin, a commonly used cancer chemotherapeutic, has a dose-limiting side effect of nephrotoxicity. Approximately 30% of patients administered cisplatin suffer from kidney injury, and there are limited treatment options for the treatment of cisplatin-induced kidney injury. Suramin, which is Federal Drug Administration-approved for the treatment of trypanosomiasis, improves kidney function after various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin-induced kidney injury. Suramin treatment before cisplatin administration reduced cisplatin-induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin-induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 h after cisplatin also improved kidney function, suggesting that the mechanism of protection is not by inhibition of tubular cisplatin uptake or its metabolism to nephrotoxic species. If suramin is to be used in the context of cancer, then it cannot prevent cisplatin-induced cytotoxicity of cancer cells. Suramin did not alter the dose-response curve of cisplatin in lung adenocarcinoma cells in vitro. In addition, suramin pretreatment of mice harboring lung adenocarcinomas did not alter the initial cytotoxic effects of cisplatin (DNA damage and apoptosis) on tumor cells. These results provide evidence that suramin has potential as a renoprotective agent for the treatment/prevention of cisplatin-induced acute kidney injury and justify future long-term preclinical studies using cotreatment of suramin and cisplatin in mouse models of cancer.
Assuntos
Injúria Renal Aguda/prevenção & controle , Cisplatino , Rim/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Suramina/farmacologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Citoproteção , Dano ao DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: It is well known that liver and lung injury can occur simultaneously during severe inflammation (e.g., multiple organ failure). However, whether these are parallel or interdependent (i.e., liver-lung axis) mechanisms is unclear. Previous studies have shown that chronic ethanol (EtOH) consumption greatly increases mortality in the setting of sepsis-induced acute lung injury (ALI). The potential contribution of subclinical liver disease in driving this effect of EtOH on the lung remains unknown. Therefore, the purpose of this study was to characterize the impact of chronic EtOH exposure on concomitant liver and lung injury. METHODS: Male mice were exposed to EtOH-containing Lieber-DeCarli diet or pair-fed control diet for 6 weeks. Some animals were administered lipopolysaccharide (LPS) 4 or 24 hours prior to sacrifice to mimic sepsis-induced ALI. Some animals received the tumor necrosis factor-alpha (TNF-α)-blocking drug, etanercept, for the duration of alcohol exposure. The expression of cytokine mRNA in lung and liver tissue was determined by quantitative PCR. Cytokine levels in the bronchoalveolar lavage fluid and plasma were determined by Luminex assay. RESULTS: As expected, the combination of EtOH and LPS caused liver injury, as indicated by significantly increased levels of the transaminases alanine aminotransferase/aspartate aminotransferase in the plasma and by changes in liver histology. In the lung, EtOH preexposure enhanced pulmonary inflammation and alveolar hemorrhage caused by LPS. These changes corresponded with unique alterations in the expression of pro-inflammatory cytokines in the liver (i.e., TNF-α) and lung (i.e., macrophage inflammatory protein-2 [MIP-2], keratinocyte chemoattractant [KC]). Systemic depletion of TNF-α (etanercept) blunted injury and the increase in MIP-2 and KC caused by the combination of EtOH and LPS in the lung. CONCLUSIONS: Chronic EtOH preexposure enhanced both liver and lung injury caused by LPS. Enhanced organ injury corresponded with unique changes in the pro-inflammatory cytokine expression profiles in the liver and the lung.
Assuntos
Etanol/farmacologia , Lesão Pulmonar/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Líquido da Lavagem Broncoalveolar/química , Quimiocina CXCL2/metabolismo , Quimiocinas/metabolismo , Etanercepte/farmacologia , Lipopolissacarídeos , Fígado/metabolismo , Lesão Pulmonar/induzido quimicamente , Masculino , Camundongos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/biossíntese , Animais , Asma/enzimologia , Asma/genética , Asma/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Inativação Gênica , Células HeLa , Humanos , Proteínas de Membrana/deficiência , Camundongos , Fenótipo , RNA Interferente Pequeno/genética , Esfingolipídeos/metabolismoRESUMO
Sphingolipids are a diverse set of structurally and metabolically related lipids that have numerous functions in cell structure and signaling. The regulation of these lipids is critical for normal cell function and disregulation has been implicated in pathophysiological conditions such as cancer and inflammation. Here we examine control of the initiating, and rate limiting, enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). We find that de novo synthesis of sphingolipid is stimulated by a number of cancer chemotherapeutics, suggesting that this may be an important aspect of their cytotoxic effects. The three ORMDL proteins are membrane proteins of the endoplasmic reticulum related to the yeast Orm proteins, which have been shown to be homeostatic regulators of SPT. We find that the ORMDL proteins are also negative regulators of SPT that transmit cellular levels of sphingolipids to SPT. The three isoforms have redundant functions in this system. The sphingosine kinases (sphingosine kinase-1 and -2) phosphorylate both sphingosine, which is released from ceramide, but also dihydrosphingosine, which is in the de novo biosynthetic pathway. We therefore examined the role of the sphingosine kinases in controlling de novo ceramide biosynthesis and find that sphingosine kinase-1 does indeed act as a negative regulator of this pathway. This establishes that sphingosine kinase, in addition to producing sphingosine-1-phosphate as a signaling molecule, also consumes dihydrosphingosine to regulate ceramide synthesis. Our studies demonstrate that there are multiple mechanisms of regulation of SPT and suggest that these regulators are important mediators of cell stress responses.
Assuntos
Ceramidas/biossíntese , Lisofosfolipídeos/biossíntese , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/análogos & derivados , Animais , Ceramidas/genética , Humanos , Lisofosfolipídeos/genética , Proteínas de Membrana/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingosina/biossíntese , Esfingosina/genética , Estresse Fisiológico/genéticaRESUMO
Histone deacetylase 6 (HDAC6) controls acetylation of a number of cytosolic proteins, most prominently tubulin. Tubacin is a small molecule inhibitor of HDAC6 selected for its selective inhibition of HDAC6 relative to other histone deacetylases. For this reason it has become a useful pharmacological tool to discern the biological functions of HDAC6 in numerous cellular processes. The interest of this laboratory is in the function and regulation of sphingolipids, a family of lipids based on the sphingosine backbone. Sphingolipid biosynthesis is initiated by the rate limiting enzyme serine palmitoyltransferase (SPT). Sphingolipids have critical and diverse functions in cell survival, apoptosis, intra- and intercellular signaling, and in membrane structure. In the course of examining the role of HDAC6 in the regulation of sphingolipid biosynthesis we observed that tubacin strongly inhibited de novo synthesis whereas HDAC6 knockdown very moderately stimulated synthesis. We resolved these seemingly contradictory results by demonstrating that, surprisingly, tubacin is a direct inhibitor of SPT activity in permeabilized cells. Furthermore tubacin inhibits de novo sphingolipid synthesis in intact cells at doses commonly used to test HDAC6 function and does so in an HDAC6-independent manner. Niltubacin is a chemical analog of tubacin which lacks tubacin's HDAC6 activity, and so is often used as a control for off-target effects of tubacin. We find that niltubacin is inactive in the inhibition of sphingolipid biosynthesis, and so does not serve to distinguish the inhibitory effects of tubacin on HDAC6 from those on sphingolipid biosynthesis. These results indicate that caution should be used in the use of tubacin to study the role of HDAC6.
Assuntos
Anilidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Esfingolipídeos/antagonistas & inibidores , Ácidos Dicarboxílicos/farmacologia , Células HeLa , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , RNA Interferente Pequeno/genética , Esfingolipídeos/biossínteseRESUMO
Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions in endothelial cells. We previously showed that S1P receptor subtype 2 (S1P2) is significantly up-regulated in the atherosclerotic endothelium (J. Biol. Chem. 283:30363, 2008). In this study, we investigated the roles of S1P2-mediated signaling in the proinflammatory responses of endothelial cells. Treatment with tumor necrosis factor-α (TNFα), a proinflammatory cytokine, increased the expression of S1P2 receptors in endothelial cells. TNFα treatment also enhanced sphingosine kinase 1 expression and increased S1P production. Pharmacological inhibition or knockdown of S1P2 receptors completely abrogated the TNFα-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression in endothelial cells. In contrast, pharmacological inhibition or knockdown of other S1P receptor subtypes had no effect on the TNFα-stimulated ICAM-1 and VCAM-1 expression. Moreover, ectopic expression of S1P2 receptors increased VCAM-1 and ICAM-1 expression in endothelial cells in response to S1P stimulation. Mechanistically, we show that antagonizing S1P2 signaling markedly inhibited the TNFα-stimulated NFκB activation. Utilizing the NFκB reporter luciferase assay, the S1P/S1P2 signaling was shown to stimulate NFκB activation. Moreover, the S1P/S1P2-stimulated VCAM-1/ICAM-1 expression was completely abolished by the pharmacological inhibitor of NFκB. Collectively, our data suggest that TNFα treatment activates autocrine S1P/S1P2 signaling, which subsequently activates NFκB and leads to the proinflammatory responses in endothelial cells.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Lisofosfolipídeos/metabolismo , NF-kappa B/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: The yeast Orm1/2 proteins regulate ceramide biosynthesis. RESULTS: Depletion of the mammalian Orm1/2 homologues, ORMDL1-3, eliminates the negative feedback of exogenous ceramide on ceramide biosynthesis in HeLa cells. CONCLUSION: ORMDL proteins are the primary regulators of ceramide biosynthesis in mammalian cells. SIGNIFICANCE: Therapeutically manipulating levels of the pro-death lipid, ceramide, requires a molecular understanding of its regulation. The mammalian ORMDL proteins are orthologues of the yeast Orm proteins (Orm1/2), which are regulators of ceramide biosynthesis. In mammalian cells, ceramide is a proapoptotic signaling sphingolipid, but it is also an obligate precursor to essential higher order sphingolipids. Therefore levels of ceramide are expected to be tightly controlled. We tested the three ORMDL isoforms for their role in homeostatically regulating ceramide biosynthesis in mammalian cells. Treatment of cells with a short chain (C6) ceramide or sphingosine resulted in a dramatic inhibition of ceramide biosynthesis. This inhibition was almost completely eliminated by ORMDL knockdown. This establishes that the ORMDL proteins mediate the feedback regulation of ceramide biosynthesis in mammalian cells. The ORMDL proteins are functionally redundant. Knockdown of all three isoforms simultaneously was required to alleviate the sphingolipid-mediated inhibition of ceramide biosynthesis. The lipid sensed by the ORMDL-mediated feedback mechanism is medium or long chain ceramide or a higher order sphingolipid. Treatment of permeabilized cells with C6-ceramide resulted in ORMDL-mediated inhibition of the rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase. This indicates that C6-ceramide inhibition requires only membrane-bound elements and does not involve diffusible proteins or small molecules. We also tested the atypical sphingomyelin synthase isoform, SMSr, for its role in the regulation of ceramide biosynthesis. This unusual enzyme has been reported to regulate ceramide levels in the endoplasmic reticulum. We were unable to detect a role for SMSr in regulating ceramide biosynthesis. We suggest that the role of SMSr may be in the regulation of downstream metabolism of ceramide.
Assuntos
Ceramidas/biossíntese , Retroalimentação Fisiológica , Proteínas de Membrana/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genéticaRESUMO
BACKGROUND: Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). RESULTS: We found that the introduction of activated H-Ras(V12) into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. CONCLUSION: Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.
Assuntos
Adenocarcinoma/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas ras/metabolismo , Adenocarcinoma/química , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Ativação Enzimática , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Consumo de Oxigênio , RNA Interferente Pequeno/genética , Transplante HeterólogoRESUMO
Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.
Assuntos
Membrana Celular/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos/metabolismo , Animais , Membrana Celular/metabolismo , Ceramidas/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Especificidade por SubstratoRESUMO
The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.
Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Células HEK293 , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingolipídeos/química , Esfingosina/metabolismoRESUMO
Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.
Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Membrana Celular/metabolismo , Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esfingosina/metabolismoRESUMO
The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.
Assuntos
Proteína Quinase CDC2/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Ciclinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Fosfofrutoquinase-2/metabolismo , Fosfatases cdc25/metabolismo , Regulação Alostérica , Proteína Quinase CDC2/genética , Núcleo Celular/genética , Ciclina D3 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Citoplasma/enzimologia , Citoplasma/genética , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Frutosefosfatos/genética , Frutosefosfatos/metabolismo , Glucose/genética , Glucose/metabolismo , Glicólise , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Fosfofrutoquinase-2/genética , Fosfatases cdc25/genéticaRESUMO
Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P(1)-mediated signaling by SEW2871 and FTY720, two agonists of S1P(1), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P(1)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P(2) signaling by JTE-013, a specific antagonist of S1P(2), S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P(1)- and S1P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P(1) and S1P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.
Assuntos
Permeabilidade Capilar/fisiologia , Lisofosfolipídeos/fisiologia , Músculo Esquelético/irrigação sanguínea , Receptores de Lisoesfingolipídeo/fisiologia , Transdução de Sinais/fisiologia , Esfingosina/análogos & derivados , Envelhecimento/fisiologia , Animais , Artérias Carótidas/fisiologia , Imunofluorescência , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Homeostase , Infusões Intra-Arteriais , Lisofosfolipídeos/administração & dosagem , Lisofosfolipídeos/farmacologia , Masculino , Músculo Esquelético/fisiologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/fisiologia , Esfingosina/administração & dosagem , Esfingosina/farmacologia , Esfingosina/fisiologiaRESUMO
Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.